The cytochrome P450 proteins are the major enzymes catalyzing the oxidation of drugs in the body, as well as steroids, carcinogens, pesticides, and numerous other compounds. Since there are at least 30 different cytochrome P450 enzymes, it is of considerable interest in the pharmaceutical and other fields to identify which of these enzymes are most important in the metabolism of individual compounds. There are now numerous examples of adverse drug-drug interactions and side effects that can now be understood in terms of the cytochrome P450 enzymes.
In many cases there are considerable differences between these enzyme systems in experimental animal models and humans, and there has been an increasing emphasis on the use of human enzymes in drug development, toxicity testing, and related fields. However, the isolation of enzymes from human tissues has been a problem because of the limited supply of tissue (esp. liver) and the difficulty involved in separation. With the availability of cDNA clones for several of these cytochrome P450s, it has become possible to express the proteins in artificial vector systems. Work with mammalian cell systems has been fairly extensive in recent years. However, these systems are expensive and the concentrations of the enzymes produced are very low. Alternative strategies involve expression in yeast and in baculovirus (insect cells). These systems are capable of producing higher concentrations of cytochrome P450 proteins but have some inherent problems. Yeast cells are very difficult to break. Baculovirus systems are sensitive to heme concentrations and also expensive to use. A presentation of articles on various systems is set forth in Methods in Enzymology, Vol. 206, Academic Press (1991).
In many respects, expression of cytochrome P450 proteins in bacteria is the most useful system because of the potentially high yields and low cost. However, the membraneous nature of the cytochrome P450 enzymes and other factors have been problematic in the use of most of the typically available vector systems. However, some progress has been made since 1990.
Larson et al. (J. Biol. Chem. 266, 7321-7324, 1991) found that rabbit cytochrome P450 2E1 could be expressed in Escherichia coli using the commercially available vector pKK233-2. In the course of these and subsequent investigations, they found that the protein was still catalytically active even if a 26-45 amino acid segment was removed. They pursued this strategy with the hope of producing a protein that would be soluble because of the deletion of the hydrophobic tail (see also Pernecky et al. Proc. Natl. Acad. Sci. 90, 2651-2655, 1993). Li and Chiang (J. Biol. Chem. 266, 19186-19191, 1991) also reported that rat cytochrome P450 7A could be produced in E. coli using a pKK vector. They changed the second codon to alanine and also found that deletion of 23 residues of the N-terminus raised the level of production. Another key study was that Barnes et al. (Proc. Natl. Acad Sci. USA 88, 5597-5601, 1991), who did not delete residues but changed the N-terminus of bovine cytochrome P450 17A to MALLLAVFL . . . (SEQ. ID. NO.: 6) and found considerable expression, using the vector called pCW (tac/tac promoter). This same sequence was inserted at the end of human cytochrome P450 1A2 to achieve high expression levels (in the pCW vector) (Fisher et al., FASEB J. 6, 759-764, 1992). Some purification of cytochrome P450s expressed in bacteria had been done (see the Larson et al. and Li and Chiang references), although numerous column steps were employed and the practicality is difficult to establish.
The inventions concerning various human cytochrome P450s provided herein are based in part on five factors: (1) some modification of the 5' terminus has been necessary in every case, (2) the presence of an alanine in the second position (GCT codon) is useful but not always sufficient, (3) the MALLLAVFL (SEQ. ID. NO.: 6) sequences (Barnes et al.) works well in some cases but not others, (4) commercially available vectors, including pKK, are not very useful, and (5) there is not a universal conclusion involving the effect of deletion of a large segment of the 5'-terminus on protein expression.
The present invention greatly improves on the state of the art by providing improved vectors for expression of various cytochrome P450 proteins. Also provided are improved strategies for the facile purification of recombinant human cytochrome P450 proteins utilizing novel combinations of detergents and enzyme inhibitors, as well as a novel purified recombinant P450 1A1, which is catalytically active.